Methods and System for Photo-activated Hydrogen Generation

ABSTRACT

Systems and methods for providing alternative fuel, in particular hydrogen photocatalytically generated by a system comprising photoactive nanoparticles and a nitrogenase cofactor are provided. In one aspect, the system includes a water soluble cadmium selenide nanoparticle (CdSe) surface capped with mercaptosuccinate (CdSe-MSA); and a NafYFeMo-co complex comprising a NafY protein and an iron-molybdenum cofactor (FeMo-co); wherein the CdSe-MSA and the NafYFeMo-co complex are present in about 1:1 molar ratio in a CdSe-MSANafYFeMo-co system. In various embodiments, when illuminated, the CdSe-MSANafYFeMo-co system is capable of photocatalytically producing hydrogen gas for an extended period of, e.g., at least 5 hours, at least 10 hours, or at least 90 hours. Methods for making and using the same are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application of U.S.application Ser. No. 14/620,386 filed Feb. 12, 2015, which claims thebenefit of and priority to U.S. Provisional Application No. 61/939,430filed Feb. 13, 2014, the entire disclosure of each of which applicationsis incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates in general to systems and methods forproviding alternative fuel, in particular hydrogen photocatalyticallygenerated by a system comprising photoactive nanoparticles and anitrogenase cofactor.

BACKGROUND

With trade and commerce of energy resources and ecological threatsstemming from their use impacting geopolitical stability, the reality isthat humankind is at a crossroads, which in turn is driving animperative to develop alternative energy sources. Humankind's tremendousindustrial and technological progress over the last two centuries hasbeen driven by the natural abundance and availability of fossil fuels.As those reserves deplete, the prudent course of action would be todevelop other readily available fuel sources. Currently, 15 terawatts(1.5×10¹³ Watts) of energy are consumed worldwide annually, with fossilfuels providing 86% of that energy. The categories of energy forms usedeach year are 40% petroleum, 23% natural gas, 23% coal, 8% nuclear, and6% from renewable sources. The projected years left in reserves at thecurrent usage levels are estimated at 47 years for petroleum reserves,60 years for natural gas reserves and 131 years for coal reserves [1].

Renewable energy sources include solar, wind, hydroelectric andgeothermal options. Solar energy is accessed and utilized in severalways. An important way is the use of silicon based solar cells thatproduces electric current. A recent important breakthrough in theresearch of inexpensive materials for energy applications is the use ofa silicon sheet coated with a cobalt based catalyst on one side and anickel-molybdenum-zinc alloy layer on the opposite side [2]. Thisdevice, named the artificial leaf, produces notable amounts of hydrogenand oxygen when submerged in water and illuminated. Another majorcurrent research effort involves the use of TiO₂ exposed to UV radiationwhich splits water into hydrogen and oxygen [3]. Water-splitting TiO₂ ismostly used in fuel cells that generate electric current from theoxidation of hydrogen. The challenge over the last three decades hasbeen to develop novel materials to perform water splitting with energyfrom the visible light region of the electro-magnetic spectrum. The useof nanoparticles alone or in composite materials with TiO₂ show promisefor improving this exploitation of solar energy [4].

Another research area toward the goal of accessing solar energy, butwith a somewhat different outcome is the production of hydrogen as analternative fuel source by utilizing biomolecules. Some researchersusing biological systems to produce hydrogen, utilize one or both of twoenzymes of hydrogenases and nitrogenases found in certainmicroorganisms. Developing ways to utilize and adapt this hydrogengenerating ability of these enzyme systems can be grouped in three broadapproaches: using an enzyme itself; forming a hybrid between differentenzyme components and a synthetic material, or by synthesizing abiomimetic analog of the biological catalyst.

Some research efforts using biomolecules involve the hydrogenases andnitrogenases with the goal of evolving hydrogen. There are severalpublished methods that have photocatalytically generated hydrogen. Forexample, self-assembled complexes between cadmium telluride nanocrystalsand Fe—Fe hydrogenase from Clostridium acetobutylicum when illuminatedwith a visible light source have produced hydrogen with ascorbic acidserving as an electron donor [5]. A NiFeSe-hydrogenase fromDesulfomicrobium baculatum complexed with ruthenium dye-sensitized TiO₂was illuminated either with a tungsten halogen lamp or with sunlight andwas effective for production of hydrogen [6]. An altered molybdenum-ironprotein (MoFe protein), component I protein of nitrogenase, wascomplexed with Ru(bypy)2 near the catalytic site. When the complex wasilluminated with a xenon/mercury lamp and provided with the substratesof protons or acetylene, the system produced the corresponding reducedproducts of hydrogen and ethylene [7].

However, the above systems have only displayed limited capability andefficiency in producing hydrogen (H₂). In particular, the longestlasting hydrogen production system utilizing inexpensive nanoparticlescomplexed to a nitrogenase or hydrogenase in published research to dateis only 4 hours. Thus, a need exists for improved systems and methodsfor producing hydrogen, in particular over an extended or prolongedperiod of time (e.g., for at least 5 hours, at least 10 hours, at least50 hours, at least 90 hours, or at least 100 hours, or longer).

SUMMARY

Systems and methods for photocataytically producing hydrogen gaspassively as an alternative fuel source are provided herein. In oneaspect, a CdSeNafYFeMo-co system has been developed, wherein cadmiumselenide nanoparticles are complexed with NafY protein containingiron-molybdenum cofactor, FeMo-co, from nitrogenase. Interrogation ofthe CdSeNafYFeMo-co system by electron paramagnectic spectroscopy(EPR) suggested electron transfer events from FeMo-co. When the systemis illuminated with visible light hydrogen gas can be evolved. TheCdSeNafYFeMo-co system unexpectedly holds up for a prolonged period oftime (e.g., 90 plus hours) while producing hydrogen throughout.

In one aspect, a system for photocatalytically producing hydrogen gas isprovided. The system includes a water soluble cadmium selenidenanoparticle (CdSe) surface capped with mercaptosuccinate (CdSe-MSA);and a NafYFeMo-co complex comprising a NafY protein and aniron-molybdenum cofactor (FeMo-co). In some embodiments the CdSe-MSA andthe NafYFeMo-co complex are present in about 1:1 molar ratio to form aCdSe-MSANafYFeMo-co system. In various embodiments, when illuminated,the CdSe-MSANafYFeMo-co system is capable of photocatalyticallyproducing hydrogen gas for an extended period of, e.g., at least 5hours, at least 10 hours, or at least 90 hours.

In some embodiments, the system can be kept under anaerobic conditionsand in the presence of a dithionite salt. The dithionite salt can be,e.g., sodium dithionite. In some embodiments, the dithionite salt iskept at a constant concentration of, e.g., 2 mM, which helps continuousand extended hydrogen production by the system. The system can also bekept at a temperature between, e.g., about 30-40° C., about 35° C., orabout 40° C., to favor formation of the monomer form of dithionite.

In another aspect, a method of producing hydrogen gas is provided. Themethod includes illuminating the CdSe-MSANafYFeMo-co system describedherein. In some embodiments, the method includes producing hydrogen gasfor an extended period of, e.g., at least 5 hours, at least 10 hours, orat least 90 hours. Fuel cells containing the CdSe-MSANafYFeMo-cosystem described herein are also provided.

In a further aspect, a method for preparing a system forphotocatalytically producing hydrogen gas is provided. The methodincludes: (a) providing a water soluble cadmium selenide nanoparticle(CdSe) surface capped with mercaptosuccinate (CdSe-MSA); (b) providing aNafYFeMo-co complex comprising a NafY protein and an iron-molybdenumcofactor (FeMo-co); and (c) mixing the CdSe-MSA and the NafYFeMo-cocomplex under anaerobic conditions to form a CdSe-MSANafYFeMo-cosystem, wherein when illuminated, the CdSe-MSANafYFeMo-co system iscapable of photocatalytically producing hydrogen gas.

In some embodiments, step (a) further comprises exchanging surfacecapping agent from trioctylphosphine (TOP) in a CdSe-TOP nanoparticle tomercaptosuccinate, to form the CdSe-MSA. In one embodiment, the CdSe-TOPnanoparticle has a diameter of about 2.4 nm to about 2.7 nm and/or theCdSe-MSA has a diameter of about 2.6 nm. In certain embodiments, theexchanging step is performed in methanol under reflux in the presence ofa base such as quaternary ammonium salts. For example, the base can betetrabutylammonium hydroxide.

In certain embodiments, in step (b), the NafY protein is derived fromAzotobacter vinelandii (wild type). The FeMo-co can be derived from amolybdenum-iron protein (MoFe protein). In one embodiment, the MoFeprotein is derived from Azotobacter vinelandii strain DJ995. Step (b)can further include combining the NafY protein with stepwise aliquots ofthe FeMo-co to form the NafYFeMo-co complex. For example, the FeMo-cocan be provided in N-methylformamide (NMF) solution and added stepwiseto the NafY protein so that NMF does not exceed about 3% (v/v).

In some embodiments, in step (c), the CdSe-MSA and the NafYFeMo-cocomplex are provided at about 1:1 molar ratio. The CdSe-MSA can beprovided in the presence of a dithionite salt. In some embodiments, thedithionite salt can be kept at a sufficiently low concentration so as toallow the CdSe-MSA and the NafYFeMo-co complex to bind to each other.Thereafter, the dithionite salt concentration can be increased (e.g., byabout 5 fold, 10 fold, 20 fold, or higher or lower) to facilitatehydrogen gas production by the CdSe-MSANafYFeMo-co system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of the Iron-Molybdenum Cofactor (FeMo-co). TheFe₇MoS₉C—(R)-homocitrate cluster is comprised of 7 irons (green), 9sulfurs (yellow), one carbon (gray), one molybdenum (magenta), and one(R)-homocitrate ligand.

FIG. 2. Representation of CdSe Nanoparticles Complexed with FeMo-co.Visible light excites CdSe enabling photoreduction of FeMo-co thatcatalyzes the production of hydrogen from available protons in solution.

FIGS. 3A-3C. CdSe-TOP Fluorescence Quenching with Added FeMo-co or NMF.(FIG. 3A) Fluorescence emission spectra of CdSe-TOP (10 μM) withsequential 1 μL additions of FeMo-co solution (60 μM stock). The plotsshow a decrease of fluorescence intensity for the CdSe-TOP. (FIG. 3B)Fluorescence spectra of CdSe-TOP with sequential additions of the samevolumes of the solvent NMF solution as the FeMo-co additions. The solidblack line is the CdSe-TOP before any additions. The first fiveadditions of NMF are shown with dotted lines showing an increasedphotoluminescence. With the additions of 6-10 μL NMF, the CdSe-TOPfluorescence is progressively quenched. (FIG. 3C) The data in panels Aand B are replotted as percent quenching to compare the quenching byFeMo-co (▴) and NMF ().

FIG. 4. CdSe-TOP Fluorescence Quenching by FeMo-co. The red trace is thecontrol fluorescence emission spectrum of CdSe-TOP without any quencher.The blue trace is fluorescence emission spectrum of CdSe-TOP quenched byaddition of the solvent NMF solution. The black line is the fluorescenceemission spectrum with addition of air oxidized FeMo-co to CdSe-TOP. Thegreen line is the emission spectrum of FeMo-co added to CdSe-TOP.

FIG. 5. CdSe-TOPFeMo-co System Investigated by EPR. EPR spectra areshown for MoFe protein (180 μM) (trace 1), FeMo-co (60 μM) (trace 2) andCdSe-TOPFeMo-co complex with CdSe-TOP (2.5 mM), FeMo-co (200 μM) (trace3). Inflection points for the EPR absorption peaks are indicated byg-values.

FIGS. 6A-6D. NafY Protein and CdSe-MSA Complex Formation Investigated byFluorescence Quenching. (FIG. 6A) NafY protein excited at 280 nm showingprogressive fluorescence quenching at 347 nm with additions of CdSe-MSAseen. There is also an emission at 532 nm that increases as moreCdSe-MSA is added. (FIG. 6B) Excitation of the same sample at 410 nm.With this emission the second emission in panel A is confirmed to befrom the CdSe-MSA. (FIG. 6C) Replot of the data in A shows percent NafYquenching versus CdSe-MSA concentration. The plot shows a saturationtype curve indicating the formation of a complex. (FIG. 6D) Replot ofthe data in FIG. 6B shows increasing photoluminescent intensity withincreasing CdSe-MSA concentration.

FIG. 7. FRET Emission in NafY-CdSe-MSA Complexed System Shown in Replotsof Data from FIGS. 6A and 6B. Excitation of the NafY samples was at 280nm. The emissions at 347 nm are NafY before (rust brown) and after(forest green) addition of 30 μL CdSe-MSA. The emission at 347 serves asthe donor for the CdSe-MSA. The forest green peak shows a secondemission that is the acceptor demonstrating FRET emission. The blue peakis the same sample with the addition of the CdSe-MSA excited at 410 nmand emitting at 532 nm. The pale green is NafY before the addition ofCdSe-MSA excited at 410 nm.

FIGS. 8A-8B. Photo-activated Time Dependent Reduction of MV²⁺ by ExcitedCdSe-MSA. (FIG. 8A) Absorbance spectra showing time dependent lightinduced reduction of methyl viologen by CdSe-MSA (0.72 μM). Methylviologen (1.47 mM) and dithiothreitol (14.7 mM) were added prior toillumination. (FIG. 8B) Replot of data in A shows time dependenceincrease in reduced methyl viologen absorption at 603 nm (720 nMCdSe-MSA) (black trace) compared to the control sample (green trace).Additionally a concentration dependent absorption of twice the amount ofCdSe-MSA is shown (1440 nM) (red trace).

FIG. 9. CdSe-MSA and NafYFeMo-co Complex Formation Investigated by EPR.EPR spectra are shown for 60 μM FeMo-co (trace 1); 200 μM NafYFeMo-co(trace 2); and CdSe-MSANafYFeMo-co (trace 3) with little change fromthe spectrum in Trace 2. CdSe-MSA (400 μM), NafYFeMo-co (200 μM).

FIG. 10. Illumination of CdSe-MSANafYFeMo-co Complex Characterized byEPR. EPR spectra are shown for CdSe-MSANafYFeMo-co (trace 1), afterillumination with a mercury lamp (trace 2) and after air exposure (trace3).

FIG. 11. Representation of a CdSe-MSANafYFeMo-co System Illuminatedwith Visible Light. NafYFeMo-co adsorbed on the surface of CdSe-MSA isphoto-reduced and catalyzes the production of hydrogen from protonsdissolved in solution.

FIGS. 12A-12B. Time Dependent H₂ Production by a 1:1CdSe-MSANaYFeMo-co System. (FIG. 12A) Hydrogen production by a 1:1CdSe-MSANafYFeMo-co system with illumination over 130 hours in fourdifferent experimental sets with the same reaction conditions. (FIG.12B) Two duplicate samples from within (FIG. 12A) with error bars.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, databaseentries, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

As used herein, the articles “a” and “an” are used herein to refer toone or to more than one (i.e., to at least one) of the grammaticalobject of the article. By way of example, “an element” means one elementor more than one element.

As used herein, the term “about” means within 20%, more preferablywithin 10% and most preferably within 5%.

The present disclosure relates to a catalytic system that can harnesssolar energy and produce an alternative fuel will be the paramountchallenge of this new century. Indeed by its end the most widely usedfossil fuel, petroleum, may have run dry. Several catalytic systems havebeen reported in scientific literature that utilizes both naturalproducts and man-made components. Some of the hybrid systems usehydrogenase, an enzyme that both reduces and oxidizes hydrogen inbacteria, either alone or in combination with photosystem I, coupled toplatinum. The goal of the present disclosure was to develop a systemthat would use a smaller catalytic natural component along with a lessexpensive light harvesting semi-conductor rather than the metalplatinum, which is of limited availability. Surprisingly, the system ofthe present disclosure displays unexpected longevity and can producehydrogen for a prolonger period of time. Methods for making and usingthe system are also provided.

CdSe Nanoparticles and FeMo-Co

Provided herein is a different approach than conventional methods forphotocatalytically generating hydrogen. The systems and methods of thepresent disclosure, in some embodiments, include cadmium selenide (CdSe)nanoparticles (NPs) which are a photoactive material responsive to lightin the visible light spectrum. CdSe NPs, also referred to as quantumdots, can be synthesized by a high temperature pyrolysis with thetargeted size of, for example, 2.4 to 2.7 nanometers in diameter. Suchnanoparticles demonstrate unique properties compared to bulk sizedparticles, such as size tunable luminescence. When synthesized they canbe dissolved in organic solvents, such as chloroform or octadecence[35].

CdSe NPs have a band gap energy near the thermodynamic potential ofhydrogen ion reduction at pH of 7. When CdSe NPs are excited with lightenergy exceeding their band gap, electrons are promoted from the valenceband to the conduction band, generating excitons, electron-hole pairs.If a charge separation can be maintained, the electrons can be availablefor transfer to an adsorbed species on the nanoparticle surface [3].

In some embodiments, a natural catalytic substance, iron-molybdenumco-factor, FeMo-co, from nitrogenase that reduces hydrogen ions to formhydrogen gas can be introduced to the surface of the cadmium selenidenanoparticle. The use of the natural product hydrogenase was mentionedpreviously as a component in other alternative fuel research methods.Instead of hydrogenase, however, the choice for a natural catalyst inthis disclosure is the iron-molybdenum co-factor in nitrogenase found indiazotrophic bacteria. It is the most studied iron-sulfur cluster of itskind among the nitrogenases. FeMo-co catalyzes the energetically uphillreduction of nitrogen gas, considered inert, to form NH₃ and hydrogen asa byproduct, thereby providing the major route by which nitrogen entersthe biosphere. It's instructive to compare the work of FeMo-co toHaber-Bosch, a man-made process that requires 200-300 atmospheres ofpressure and 300° C. and upwards to accomplish nitrogen fixation.Nitrogenase performs the very same with the aid of FeMo-co in ambientpressure and temperatures.

FeMo-co is shown in FIG. 1, which is the catalytic cofactor found in theactive site of the molybdenum-iron protein (MoFe protein) component ofnitrogenase. The MoFe protein and Fe protein, also denoted as componentI and component II, respectively, are the two proteins that comprisenitrogenase. The Fe protein is a 63 kDa homodimer that has a singleFe₄S₄ cluster at the dimer interface. The MoFe protein is a 240 kDaα₂β₂-heterotetramer with two types of Fe—S clusters, the P-cluster andFeMo-co. The P-cluster, a Fe₈S₇ cluster, is at the interface of the aand βsubunits of the MoFe protein. At the nitrogenase active site, theiron-molybdenum cofactor (FeMo-co) catalyzes the reduction of nitrogenand protons to form ammonia and hydrogen. FeMo-co is aFe₇MoS₉C—R-homocitrate cluster (FIG. 1) within the active site in theα-subunits. Each FeMo-co is conjugated at the terminal iron by athiolate of a cysteine (α-275^(Cys)) and at the other, end on themolybdenum by the ε-N of a histidine (α-442^(His)) [8, 9].The-R-homocitrate is coordinated through its C-2 carboxyl and hydroxylgroups to the molybdenum [10]. The Fe-protein binds Mg-ATP, docks to theMoFe protein, hydrolyzes the bound Mg-ATP, and couples the liberatedfree energy to inter-protein transfer of a single electron to theP-cluster of the MoFe protein (Equation 1). Electrons are thentransferred from the P-cluster to FeMo-co where an eight electronreduction of N₂ and protons occur producing NH₃ and H₂ (Equation 1)[11]. In the absence of any substrates, such as nitrogen or acetylene,FeMo-co readily reduces protons to form hydrogen [12, 13].

N₂+8e ⁻+16MgATP+8H⁺→2NH₃+H₂+16MgADP+16P_(i)  (Equation 1)

The best model in literature, the Thorneley-Lowe mechanism [64],describes how the Fe protein with two bound Mg-ATP binds to MoFeprotein, thought to induce a conformational change resulting in loweredactivation energy. The two MgATP undergo hydrolysis and one electron isdelivered first to the P-cluster and then to FeMo-co. The Fe proteindissociates from the Mo—Fe protein. Each of these electron transferevents are labeled E_(n), E₁₋₈, in the Thorneley-Lowe model and fourelectrons must interact with FeMo-co before the nitrogen substratebinds. An alternating pathway versus a distal pathway is now favored andproposes that at FeMo-co the two nitrogens bound to Fe are hydrogenatedalternately. After four steps, it is believed that a hydrazineintermediate forms and after step five the first NH₃ is released.Substrates, NH═NH, and NH═N—CH₃ along with trapped intermediates andgenetically modified MoFe protein allowed for this selection versus thedistal pathway which hypothetically yields different intermediates. Thesecond NH₃ is released after E₈ step [65]. In the absence of anysubstrates, such as dinitrogen or acetylene, FeMo-co readily reducesprotons to form hydrogen, done so by the formation of hydrides on Fe[66]. When nitrogen and other substrates are absent, yet provided with asource of hydrogen ions, FeMo-co readily reduces H⁺ to form hydrogen gas[67]. Thus, FeMo-co, one of the most reductive catalysts in nature, is apowerful reductant for the formation of hydrogen gas.

FeMo-co can be prepared by in vitro and/or in vivo methods such aschemical synthesis or recombinant proteins. For example, Azotobactervinelandii cells (wild-type or engineered) can be grown, harvested andstored to extract FeMo-co. In some embodiments, the genes in the cellsthat encode for the synthesis of nitrogenase within that organism can begenetically modified to include a tag (e.g., string of histidine aminoacids) fused to an end. Once expressed, the tag can facilitate theseparation and purification of the component I protein of nitrogenaseutilizing, e.g., liquid chromatography. In one example, adequate numbersof Azotobacter vinelandii bacterial cells are grown in a 150 literfermenter and harvested in a 2 day procedure. Thereafter, in a 12 to 16hour procedure, MoFe protein, component I of nitrogenase, is purifiedunder oxygen free conditions. In an additional procedure, FeMo-co canthen be extracted into N-methylformamide, an organic solvent, understrict anaerobic conditions.

Other host cells such as Escherichia coli can also be used forheterologous expression of FeMo-co. In some embodiments, using FeMo-coextracted from Component I protein from nitrogenase may be desirablesince it is homologously expressed in Azotobacter vinelandii. Homologousexpression in A vinelandii cells can, in some instances, provide largeramounts of FeMo-co in respect to the amount of hydrogenase yielded fromthe protein purification from heterologous expression.

CdSeFeMo-co Complex

Toward the goal of developing a novel advanced material forphotocatalytic H₂ production, a complex containing FeMo-co and CdSe, aphotoreductive nanoparticle is provided by the present disclosure. Sucha CdSeFeMo-co system is a passive way to produce hydrogen as analternative fuel source. The CdSeFeMo-co system couldphotocatalytically generate H₂ is substantiated by the fact that CdSenanoparticles are able to reduce methyl viologen (MV²⁺) byphotoactivation. The CdSeMV²⁺ system was illuminated and causedreduction of the MV²⁺ where the reduced state was observed by transientabsorption spectroscopy [14]. The reduction potentials of MV²⁺ andFeMo-co are very close (−0.460 mV and −0.465 mV, respectively) [15, 16].Therefore, CdSe can photo-reduce FeMo-co in the same way that it reducesMV²⁺. With illumination of a CdSeFeMo-co system, CdSe photo-reducesFeMo-co and then FeMo-co catalyzes the reduction of dissolved protons toevolve hydrogen (FIG. 2).

In certain embodiments, it is described herein how CdSe and FeMo-co arecomplexed, with and without a protein, in organic and aqueous media, andare used for generation of hydrogen. Because of the readily availablesource of solar energy, the generation of hydrogen throughphoto-catalysis represents a promising option to secure a sustainableenergy source for the future.

In some embodiments, aqueous CdSeFeMo-co may be advantageous over thatin an organic solvent. For example, the presence of an organic solventmay limit the use or compatibility with most protein applications, orapplications involving other biomolecules. Thus, in one embodiment,water soluble CdSe nanoparticles and FeMo-co are first prepared, andthen mixed together under proper conditions to form a water solublecomplex.

Water soluble CdSe nanoparticles are provided by the present disclosure.It should be noted that numerous attempts at synthesizing aqueous basedCdSe particles have failed previously, since they were not found to bephoto-luminescent and therefore not photo-active. It was only throughexhausting numerous synthetic strategies that the present disclosureidentified a suitable method of making photo-active aqueous soluble CdSenanoparticles. In some embodiments, synthesizing Water soluble CdSenanoparticles includes first synthesizing CdSe nanoparticle surfacecapped with trioctylphosphine (TOP), and exchanging the surface cappingagent with mercaptosuccinate to form the CdSe-MSA. In certainembodiments, the exchanging step is performed in methanol under refluxin the presence of a base such as tetrabutylammonium hydroxide.

In certain embodiments, to introduce FeMo-co into an aqueous system, achaperone protein can be used. In one embodiment, NafY (nitrogenaccessory factor Y), a FeMo-co insertase [60] is used to bind toFeMo-co. NafY protein is a 26 kDa chaperone protein that assists withinsertion of completely assembled FeMo-co into the apo-MoFe protein. Ithas a high affinity for FeMo-co. From mutagenesis studies, His121 inNafY is suggested to bind FeMo-co, which is located on the surface ofNafY [61]. NafY binds independently to FeMo-co or to the apo-MoFeprotein (NifDK). The presence of NafY has been observed to stabilizeapo-NifDK, and thereby prepares apo-NifDK for FeMo-co insertion [61].After the insertion of FeMo-co into apo-NifDK, the NafY dissociates fromthe activated NifDK [62].

To assemble the CdSeFeMo-co complexes of the present disclosure, anordered procedure should be followed. In one example, the procedureincludes: (a) providing a water soluble cadmium selenide nanoparticle(CdSe) surface capped with mercaptosuccinate (CdSe-MSA); (b) providing aNafYFeMo-co complex comprising a NafY protein and an iron-molybdenumcofactor (FeMo-co); and (c) mixing the CdSe-MSA and the NafYFeMo-cocomplex under anaerobic conditions to form a CdSe-MSANafYFeMo-cosystem.

In some embodiments, photo-active CdSe nanoparticles that are in aparticular size regimen (e.g., 2.4-2.7 nm) should be used. The sampleconcentration of both the CdSe nanoparticles and the FeMo-co can also becontrolled (e.g., in a 1:1 molar ratio). Anaerobic conditions throughoutthe complex preparation are important. In addition, FeMo-co inN-methylformamide (NMF) solvent be added in a stepwise manner so as notto exceed 3% v/v NMF/NafY aqueous solvent during the process. This is toprevent the NMF from degrading the NafY protein. NafY has a highaffinity for FeMo-co and during the additions of FeMo-co/NMF aliquots tothe NafY solution, FeMo-co will quickly bind to the NafY. Once bound toNafY the FeMo-co is sequestered inside NafY. This is important so as notto expose the FeMo-co to aqueous solution and thereby hydrolyze theFeMo-co. The maintenance of strict anaerobic conditions facilitates thiscoupling. The next step is to add the NafYFeMo-co solution to theCdSe-MSA. When mixing CdSe-MSA and the NafYFeMo-co, the addition ofCdSe-MSA should not be performed before the dithionite concentration isincreased so as to avoid quenching of the CdSe photoluminescence by thedithionite.

When combining CdSe NPs with FeMo-co, several considerations must begiven. FeMo-co has a standard reduction potential of −465 mV. Methylviologen's reduction potential has a similar value of −460 mV. It wasreported that adsorbed methyl viologen was reduced by photoactivation ofcadmium selenide, interrogated by transient absorption spectroscopy[63]. If FeMo-co could be introduced to cadmium selenide nano-particlesand adsorb on the surface then once the cadmium selenide is illuminatedand emitted electrons, then some novel chemistry could result. It wasfirst necessary to establish that by adding the FeMo-co to a solution ofCdSe, the FeMo-co would adsorb onto the surface of the CdSe. This can bedemonstrated by showing that there is an interaction between the two.

Several steps were first carried out to prepare the reactants before theinteraction could be demonstrated. First, CdSe nanoparticles weresynthesized by high temperature pyrolysis. The CdSe quantum dots werecapped with trioctylphosphine and solubilized in octadecene.Characterization showed that the dots were 2.4 nm in diameter and thatthey were very photoluminescent. Next, the CdSe needed to be aqueoussoluble so a ligand exchange was carried out. This was done by a refluxin methanol reaction that facilitated the exchange of mercaptosuccinicacid for the trioctylphosphine on the surface of the CdSe nanoparticle.Characterization demonstrated that the CdSe nanoparticles were now 2.6nm in diameter and that the photoluminescent property was slightlydiminished; however, the nanoparticles did retain adequatephotoluminescence to conduct further experiments.

Specifically, the photo-activity of the mercaptosuccinic capped CdSenanoparticles was demonstrated by experiments with light drivenreduction of adsorbed methyl viologen. Methyl viologen was added to CdSeand then over 30 second time intervals, UV-visible spectroscopy wasmeasured with a time dependent absorbance increase at 605 nm (FIG. 8A)As more methyl viologen was reduced by the electron transfer from thelight excited CdSe nanoparticle emission of electrons, there was acorresponding observable increase of blue colored solution. Performingligand exchange on trioctylphosphine capped nanoparticles is known todiminish photo-activity of quantum dots; however, as the fluorescencecharacterization, FRET spectra and reduction of adsorbed methyl viologendemonstrations showed, the aqueous solubilized CdSe nanoparticles wereadequately photo-active.

Demonstrating that there was an interaction between CdSe and NafYprotein utilized the tryptophan fluorescence inherent within polypeptidechains. Adding CdSe capped with mercaptosuccinic acid in aliquotsdemonstrated a concentration dependent quenching of the NafY proteinfluorescence when exciting the sample at 280 nm (FIG. 6A). Interactionbetween CdSe and NafY was further demonstrated by Forster ResonanceEnergy Transfer (FRET) when exciting the NafY and CdSe samples at 280 nmand 410 nm (FIG. 7). The donor emission of NafY fluorescence emissionexcited the CdSe which served as the acceptor. The emission at 525 nmwas that verified of CdSe confirmed by excitation of the sample at 410nm and showing the same emission peak.

Electron transfer between the CdSe and the adsorbed NafYFeMo-co wasinterrogated by the use of electron paramagnetic resonance spectroscopy(EPR). Samples of CdSeNafYFeMo-co prepared in 2.0 mM sodium dithonite25 mM Tris solution, pH 8.0, were made. The first set of EPR spectrashowed that the NafYFeMo-co system was intact after its addition andadsorption to the CdSe nanoparticles (FIG. 9). The CdSeNafYFeMo-cosample was thawed, subjected to ten seconds of intense illumination andthen flash frozen. A second EPR was taken and showed an EPR silent statethat was indicative of an electronic change. The light had driven anelectron transfer to the FeMo-co indicated by a change in the oxidationstate of the FeMo-co; the S=3/2 resting FeMo-co spin state had convertedto a spin silent state. The electron could then be available for protonreduction (FIG. 10).

In various embodiments, the CdSeFeMo-co complexes of the presentdisclosure are capable of photocatalytically producing hydrogen whenilluminated. Hydrogen generation experiments were initially set up withvarious CdSe to NafYFeMo-co ratios in 25 mM Tris, pH 8.0, 2 mM sodiumdithionite. In some embodiments, 1:1 CdSe:NafYFeMo-co can be used. Thepurpose of the dithionite in excess was twofold. It served to preservethe anaerobicity of the sample and was a sacrificial electron donor tofill the electron hole generated by exciton creation by light energy.The samples were set up on a Peltier cooling device and exposed to a 500watt hydrogen lamp. The temperature was at 28-30° C. The headgas wasdrawn and injected into gas chromatograph to detect hydrogen gasgeneration which continued until approximately 114 hours after sampleset up. In four different experimental sets under the same reactionconditions, hydrogen was produced with a rate of 105.3 moles of H₂/moleof CdSeNafYFeMo-co (FIG. 12A). The rate could also be expressed as0.069 μmol H₂/mg protein/hour. This is comparable to rates generated byother PSI/Pt/hydrogenase systems [68].

Some of the advantages of using CdSe versus some other photoactivematerials include the band gap energy of CdSe and its ability to emitelectrons when excited by light with the energy that isthermodynamically matched to the iron-molybdenum cofactor (FeMo-co) andits ability to reduce protons. The FeMo-co, in some embodiments, can becoordinated to its natural chaperone protein, NafY (26 kDa), which isless than half the size of the hydrogenases used in other hybridsystems. FeMo-co can be stabilized in NafY, which is an improvement overits extracted state, dissolved in N-methylformamide. Once chargeseparation is achieved versus the electron's tendency to relax back tothe ground state, the close proximity to the surface of the CdSefacilitates more efficient electron transfer to the FeMo-co.Furthermore, the CdSe band gap energy is not too oxidative once theelectron hole is generated as compared to TiO₂, another photoactivematerial used in some hybrid systems. TiO₂ tends to both oxidize aminoacids within the polypeptide chain of the enzyme and also may oxidizewater to form O₂ which would jeopardize the integrity of theNafYFeMo-co. The CdSeFeMo-co system also avoids the use of theexpensive and rarely found platinum, used in many of the hybrid systemsin literature where platinum is at risk to be poisoned by environmentalcontaminants and be rendered inactive.

Furthermore, it has been surprisingly found that a CdSeNafYFeMo-cosystem can produce hydrogen for a prolonged period of time (e.g., for atleast 5 hours, at least 10 hours, at least 50 hours, or at least 90hours, or longer) compared to the published systems which producehydrogen for up to 4 hours at the most. In one example, theCdSeNafYFeMo-co system is operational for 114 hours, whereas otherconventional systems were only operational for minutes or for a fewhours.

Additional variables can be optimized to improve hydrogen productionyield, rate and/or longevity. pH is probably the first. pH was initiallyat 8.0, the pH of the Tris buffer solution. The pH of the buffer can bereasonably lowered and thus provide more H⁺ for reduction. In addition,the system can withstand warmer temperatures and this can help inshifting the equilibrium towards the formation of the SO₂ ⁻, the radicalmonomer form of S₂O₄ ²⁻, the real reactive species in the dithionite. Itwas found that in a temperature range of 2-40° C., the monomer form isfavored at higher temperatures. Furthermore, dithionite concentrationcan be increased. Finally, a slight overpotential can be provided if thesystem is incorporated to a fuel cell. It is possible to exploredepositing the CdSeNafYFeMo-co on a graphite electrode so thatdiffusion rates are not a limiting factor. Thus, the system demonstrateda stability and longevity that can be extended to make it adaptive forcommercial applications.

In conclusion, there are many advantages to a CdSeNafYFeMo-co systemcompared to other hydrogen generation systems in the literature, suchas:

-   -   CdSe nanoparticles are relatively inexpensive to make and are        reliably photo-active. They are stable for long periods. This is        in contrast to the expensive catalyst, platinum, which is of        limited availability, and is used in numerous other hybrid        systems in research. Platinum is easily poisoned by        environmental contaminants.    -   CdSe, if provided with an adequate sacrificial electron donor is        stable for long periods of time and withstands photo-degradation        characteristic of other semiconductor systems such as CdS.    -   The energy required to produce hydrogen is a good thermodynamic        match for the electrons emitted from CdSe.    -   FeMo-co is one of the most powerful catalysts in nature. It        catalyzes the production of ammonia and hydrogen in ambient        temperatures and pressures. In contrast, man-made catalysis to        produce ammonia requires 450° C. and 200 atmospheres of        pressure. In the absence of nitrogen, FeMo-co will catalyze the        production of hydrogen gas as in this water based system in        ambient temperatures and pressure.    -   In the CdSeNafYFeMo-co system of the present disclosure,        FeMo-co is bound to NafY (26 kDa), less than one half the size        protein compared to hydrogenase (56 kDa) used in similar        systems. NafY protein is the chaperone protein which is active        to insert FeMo-co into the nitrogenase component protein after        synthesis occurs. NafYFeMo-co is stable, yet accessible for        electron transfer from the photo-active CdSe. Efficient electron        transfer reactions are all about the proximity of reactive        components.    -   Similar systems, one using hydrogenase and another with a        component protein of nitrogenase, produced hydrogen for five        minutes and 50 minutes, respectively. There are other systems        using photosystem I as a light harvesting component combined        with either platinum or hydrogenase which lasted for longer        periods, but use the expensive platinum material. The        CdSeNafYFeMo-co system of the present disclosure produced        hydrogen for, e.g., 100 plus hours and has the potential to be        optimized to perform for longer periods.    -   The hydrogen produced by CdSeNafYFeMo-co system is pure versus        hydrogen produced by steam reforming. Impurities in hydrogen        streams are undesirable since they can poison expensive metal        catalysts such as platinum in fuel cells.    -   A practical application of the CdSeNafYFeMo-co system is to        incorporate it with a fuel cell. As a result, fuel cells can be        provided with a continuous flow of hydrogen from the        CdSeNafYFeMo-co system to react it with oxygen from the air to        enable production of electric current.

Examples

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

1. Introduction

In the following examples, CdSe and FeMo-co were complexed with andwithout a protein, in organic and aqueous media, and were used forgeneration of hydrogen. First, to prove that a complex would form,fluorescence quenching was used to interrogate complex formation betweenchloroform soluble CdSe nanoparticles and FeMo-co, as well as betweenaqueous soluble CdSe and NafY protein and FeMo-co. Next, to show thatelectron transfer would occur, electron paramagnetic resonancespectroscopy was used. The complex with the most promise for hydrogengeneration was the CdSe-MSANafYFeMo-co system, which when illuminatedwith visible light evolved hydrogen consistently and for prolongedperiods of time compared to published systems. The CdSeFeMo-co systemmay prove to be a promising alternative fuel source.

2. Materials and Methods 2.1 UV-Vis Absorbance and Fluorescence EmissionSpectroscopy

UV-visible absorbance spectra were acquired on a Perkin-Elmer Lambda750S spectrophotometer. Fluorescence emission was measured using aHoriba Jobin Yvon Fluoromax 4 Spectrofluorometer at excitation slitwidths of 2 or 3 nm and emission slit widths of 2 or 3 nm.

2.2 Synthesis of CdSe-TOP Nanoparticles

CdSe-TOP quantum dots with particle sizes of 2.4 to 2.7 nm weresynthesized using a high temperature pyrolysis method [14]. The CdSenanoparticles with the capping agent, trioctylphosphine (TOP), weresolubilized in chloroform and prepared according to published methods[17, 18]. The nanoparticles were stored anaerobically in the dark at 4°C. The quantum dots were characterized by UV-vis absorbance andfluorescence emission spectroscopy. The CdSe nanoparticle size wasdetermined by noting the first absorption peak, using the publishedmethod relating absorbance to nanoparticle diameter. The calculatedextinction coefficient based on the particle diameter was 59,598 M⁻¹cm⁻¹that enabled the determination of CdSe nanoparticle concentration [19].Absorbance (λ_(max)), was 515 nm and fluorescence emission (Em_(max)),when excited at 410 nm was 531 nm [20]. CdSe-TOP will be used todesignate TOP capped CdSe that is soluble in organic solvents.

2.3 Purifications of MoFe Protein, NafY, and Extraction of FeMo-co

MoFe protein was purified from Azotobacter vinelandii strain DJ995,expressed with a polyhistidine tag fused on the C terminus of nifD, aspreviously described [21]. The host cell can also be engineered toexpress NifD fused to other tags known in the art to facilitatepurification of the fusion protein. NifD is the α-subunit of MoFeprotein (NifDK). MoFe protein purity was judged to be 95% pure usingSDS-PAGE [22], stained with Coomassie blue. Quantification was performedby Biuret assay [23]. FeMo-co was acid extracted into N-methylformamide(NMF) solution using previously published methods [24], and quantifiedby assaying Fe concentration as previously described [25]. The degassedNMF solution was adjusted to a pH of 8.0 with triethylamine and sodiumdithionite was anaerobically added to a final concentration of 1 mM. Toassess FeMo-co activity, FeMo-co was added to an apo-form of the MoFeprotein to form the holo-MoFe protein and assayed by measuring hydrogenevolution activity [26].

NafY was cloned from Azotobacter vinelandii (wild type) and expressed inEscherichia coli and purified according to previously publishedprocedures [27]. Protein purity was assessed by SDS-PAGE andquantification was performed by the bicinchoninic acid assay methodusing bovine serum albumin as a standard [28].

2.4 Assembly of Complexes Between CdSe-TOP and FeMo-co

All sample preparations were performed in anaerobic septum sealedcuvettes or vials, using standard Schlenk line techniques or performedin an anaerobic chamber with 5% (v/v) hydrogen/nitrogen and less than 1ppm O₂. Gas tight syringes were used to perform liquid transfers for allexperiments.

2.5 X-Band EPR Spectroscopy

X-band EPR spectra were recorded on a Bruker Elexsys E580 X-bandspectrometer equipped with a Bruker standard rectangular TE102 resonatorand fitted with an Oxford Instruments ESR900 helium flow cryostat.Modulation frequency was set at 100 kHz and modulation amplitude was setat 1.00 mT (10.0 G). The microwave frequency was approximately 9.45 GHzwith the exact frequency noted for each spectrum and used forcalculation of g values. All spectra were recorded at 5 K using amicrowave power of 2.0 mW. Each trace was a sum of five scans unlessstated otherwise.

2.6 Assembly of CdSe-MSANafY-FeMo-co Complexes

Water soluble CdSe nanoparticles were synthesized following a publishedprocedure with some modifications. CdSe-TOP nanoparticles weresynthesized by high temperature pyrolysis and dissolved in octadecene[30]. Characterization was performed by UV-vis absorbance (λ_(max)−515nm), and fluorescence emission (Em_(max) −532 nm when excited at 410nm). Size determination of the quantum dots was 2.4 nm diameter and thecalculated extinction coefficient was 59,598 M⁻¹ cm⁻¹ [20]. To exchangethe surface capping agent from trioctylphosphine to the aqueouslysoluble mercaptosuccinate, a reaction in methanol was performed underreflux at pH of 10. Fourfold molar excess of the quaternary base,tetrabutylammonium hydroxide, as compared to the added moles ofmercaptosuccinate was used to fully deprotonate the mercaptosuccinicacid. The use of a solution of the base dissolved in methanol (40% w/v)was essential to avoid introducing any unnecessary water into thesystem.

During the work-up steps, the water soluble quantum dots wereprecipitated on the inner surface of the round bottom flask using arotary evaporator, instead of precipitation by centrifugation asdescribed in the published procedure. The quantum dots inside the flaskwere dried under vacuum overnight and resolubilized in aqueous 25 mMTris, pH 8.0, the following day. The mercaptosuccinate functionalizedCdSe quantum dots dissolved in 25 Mm Tris solution had an extinctioncoefficient of 73,681 M⁻¹ cm⁻¹[20]. Their diameter was calculated to be2.6 nm with λ_(max) at 525 nm and an Em_(max) at 538 nm. CdSe-MSA willbe used to designate the mercaptosuccinate capped water soluble quantumdots.

All samples with CdSe-MSA were set up in septum sealed cuvettes or vialsusing standard Schlenk line techniques or in an anaerobic chamber with5% (v/v) hydrogen/nitrogen with less than 1 ppm O₂. Gas tight syringeswere used to perform liquid and gaseous transfers in all theexperiments.

A reaction between CdSe-MSA and methyl viologen was performed withaqueous soluble CdSe-MSA (720 nM) combined with methyl viologen (1.47mM) and dithiothreitol (14.7 mM), with adaptations to a published method[30]. The samples were illuminated for 30 second intervals followed byUV-vis absorbance for a total time of eight minutes.

Preparation of the NafYFeMo-co complex was performed using amodification of previously described procedures [31]. NafY in 25 mM Trisand FeMo-co in NMF solution were combined with stepwise aliquots of theFeMo-co such that the NMF solution did not exceed 3% (v/v) NMF so as toavoid denaturing of the protein.

2.7 Photo-Catalyzed Hydrogen Production

CdSe-MSA and NafYFeMo-co were mixed together in 3.0 mL anaerobiccrimped seal vials in a total reaction volume of 1.5 mL, 25 mM Tris atpH 8.0, 0.2 mM Na₂S₂O₄, 2 μM to 16 μM CdSe-MSA nanoparticles and 2 μMNafYFeMo-co.

The dithionite concentration was initially kept at 0.2 mM so as to allowthe CdSe-MSA and NafYFeMo-co to bind to each other with minimalinterference from the dithionite. After one minute of mixing, theNa₂S₂O₄ concentration was increased to 2.0 mM. Prior to illumination,samples were thoroughly degassed and exchanged into argon by multiplerounds of vacuum and 3 psi of O₂ free argon. Reaction samples for thereduction of methyl viologen or hydrogen production experiments wereilluminated using a 500 Watt halogen lamp. Samples were kept 8 cm fromthe light source. The samples were continuously illuminated and thetemperature was kept at 30±2° C. Illumination times are indicated inrelevant figure legends.

Assay for the evolution of hydrogen was performed by sampling (250 μL)the headgas of reaction vials using a gas-tight syringe and analyzing ona Hewlett Packard Series II 5890 Gas Chromatograph equipped with aRestek 13× 60/80 molecular sieve column and a thermal conductivitydetector. Argon flow was set at 5.0 mL/min. Hydrogen peaks werestandardized using quantitative hydrogen standards prepared in crimpedseal vials of the same size and same headgas volume as the samples with1.5 mL water volume according to published procedures [32].

3. Results

3.1 Assembly of CdSe-TOPFeMo-co complex

The intent for forming a CdSe-TOPFeMo-co complex was to take advantageof the CdSe-TOP's photocatalytic activity and FeMo-co's ability tocatalyze H⁺ reduction. For the two components to work together andutilize solar energy to facilitate electron transfer for hydrogenproduction, the formation of a stable complex between CdSe-TOP andFeMo-co would be required.

CdSe-TOP nanoparticles are characteristically photoluminescent. Hencefluorescence emission may be used to interrogate interactions betweenCdSe-TOP and other chemical species [33]. Complex formation with anotherspecies may result in static quenching of the CdSe-TOP fluorescenceemission. Static quenching may be observed if the interaction betweendifferent components allows for overlap of molecular orbitals [34].Quenching of the CdSe-TOP fluorescence emission was used to determine ifthe added FeMo-co was bound to the surface of the nanoparticle.

The CdSe-TOPFeMo-co system has some advantages as compared to CdSe inaqueous solvents. CdSe-TOP nanoparticles in organic solvents havesignificantly greater photo-activity as compared to the same sized CdSenanoparticles in aqueous media (40 times more photoluminescence).CdSe-TOP nanoparticles exhibit the photo-activity due to their intrinsicquantum confinement characteristic. The quantization effects areobserved because their excitons, excited electrons and their electronholes are confined within small regions of space. Although quantum dotsare comprised of approximately 100 binary units of CdSe in threedimensional space, they are still small enough to exhibit the quantumproperties of a single CdSe unit [35]. Quantum confinement alsocontributes to the observed property of nanoparticle color, i.e. theenergy for excitation corresponding to the bandgap, which can be tunedby adjusting nanoparticle size within the parameters of its Bohr excitonradius (5.6 nm for CdSe) [36]. The size of the CdSe-TOP nanoparticleswas dictated and controlled by managing reaction conditions such astemperature and time during its synthesis.

Another advantage to the CdSe-TOPFeMo-co system was the ability to usechloroform as a solvent and its miscibility with NMF solution, which isused to extract FeMo-co from the MoFe protein. In addition, the NMFsolution contains 1 mM sodium dithionite. The sodium dithionite servestwo purposes in the CdSe-TOPFeMo-co system of being an oxygen scavengerand as a source of electrons [26]. FeMo-co is very oxygen sensitivehaving an approximate half-life of about 30 seconds after air exposure[26], and the sodium dithionite prevents oxidative degradation ofFeMo-co. When light excites CdSe-TOP, an electron is excited to a higherenergy state moving into the conduction band. This generates an electronhole in CdSe's valence band. The dithionite acts as a sacrificial donorto quench the electron hole [37].

As described earlier, fluorescence emission was used to probe theinteraction between CdSe-TOP and FeMo-co. Incremental 1 μL additions ofFeMo-co (60 μM stock) were made to a 10 μM solution of CdSe-TOPnanoparticles and the sample was inspected by both UV-vis absorbance andfluorescence emission spectroscopies after each addition. With theaddition of either the FeMo-co or solvent NMF solution, the UV-visabsorbance (data not shown) did not change significantly, indicatingthat the integrity of the CdSe nanoparticles in solution was intact. Incontrast, significant changes were observed in the fluorescence emissionof CdSe-TOP. Progressive addition of FeMo-co to the CdSe-TOP solutionresulted in the quenching of the CdSe-TOP fluorescence emission (FIGS.3A and 3C).

A similar titration was performed using the solvent NMF solution usedfor the extraction of FeMo-co to serve as a control (FIGS. 3B and 3C).The addition of NMF to the CdSe-TOP solution enhances its fluorescenceintensity with the addition of the first 5 μL. This effect to enhancethe fluorescence has been reported before [38]. The fluorescenceintensity does decrease with the further addition, 6 to 10 μL, of thesolvent NMF solution (FIG. 3B).

The degree of emission quenching with respect to the volume of eitherFeMo-co or NMF solution added (FIG. 3C), clearly shows a differencebetween the additions of FeMo-co solution and the additions of NMFsolution. The addition of only 5 μL of FeMo-co (60 μM in solution) wassufficient to quench 90% of the CdSe-TOP fluorescence emission. Incomparison, only 10% of the fluorescence emission was quenched with theaddition of an equivalent volume of the solvent NMF solution.Furthermore, quenching of the CdSe-TOP fluorescence by FeMo-co seems tosaturate, somewhat suggesting a specific interaction. These dataindicate that the CdSe-TOP nanoparticles and the FeMo-co do form acomplex. Additionally, the CdSe-TOP quantum dots that were mostefficient for forming a complex were in the size range of 2.4 to 2.7 nmdiameter.

Despite the demonstrated fluorescence quenching of CdSe-TOP by theaddition of FeMo-co, which suggests the formation of a complex, aquestion persisted whether the quenching is due to binding of FeMo-cothat is structurally intact. To test this, air-oxidized FeMo-co wasadded to CdSe-TOP. If the FeMo-co had degraded during the formation ofthe complex, quenching of the CdSe-TOP fluorescence would not be likely.

Three samples of CdSe-TOP (10 μM) were inspected by UV-vis absorbanceand fluorescence emission before and after six μL additions of 1) 60 μMFeMo-co, 2) air exposed degraded FeMo-co of the same concentration, or3) solvent NMF solution. Addition of FeMo-co quenched 88% offluorescence from CdSe-TOP (FIG. 4 and Table 1), and addition of thesolvent NMF solution caused 52% quenching, which represents a similardegree of fluorescence quenching in the titration experiments above.Significantly, the addition of degraded FeMo-co sample showed 61% ofquenching, which is comparable to the extent of quenching observed withaddition of the solvent NMF solution. Since the air exposed degradedFeMo-co was in NMF solution, the limited fluorescence quenching observedwith the degraded Fe^(2+/3+) was close to the fluorescence quenchingobserved with the addition of the same amount of NMF solution. Thus,structurally intact FeMo-co is binding to CdSe-TOP nanoparticles,causing CdSe-TOP fluorescence quenching. The fluorescence quenchingobserved is not due to potential dissolved Fe^(2+/3+) in solution fromdegraded FeMo-co.

TABLE 1 Percent Quenching of CdSe-TOP Fluorescence by FeMo-co Additionsto CdSe-TOP Percent Quenching* NMF, 0.1 mM dithionite 51.7 OxidizedFeMo-co 60.7 Active FeMo-co (0.36 μM) 87.7 *CdSe-TOP fluorescenceemission without additions is 518,000 arbitrary units. Percent quenchingis a measurement of CdSe-TOP fluorescence after addition of reagentssubtracted from fluorescence without the additions divided byfluorescence without additions times 100.

3.2 EPR Characterization of CdSe-TOP-FeMo-co Complex

EPR spectroscopic analysis of Fe—S clusters allows for investigation ofelectronic structures, electronic spin states and configurations.Changes observed in characteristic signals can possibly indicate adifferent electronic spin state or configuration. Oxidation statechanges can also be observed after electron transfers [39]. FeMo-co hasa characteristic S=3/2 spin state signal in its dithionite reducedresting state and also an analogous signal in the dithionite reducedholo-enzyme [40]. The CdSe-TOP nanoparticles show no EPR signal.

The fluorescence emission characterization of CdSe-TOP with addedFeMo-co had suggested that CdSe-TOPFeMo-co had formed a complex due tothe quenching of fluorescence emission of the CdSe-TOP. Withillumination of the CdSe-TOP, excited electrons could be transferred tothe FeMo-co adsorbed on the CdSe-TOP surface with a potentialobservation of a change in the FeMo-co EPR signal due to an alterationin its electronic structure.

To achieve concentrations appropriate for EPR experiments (>100 μM ofEPR active species), a much higher concentration of the CdSe-TOPnanoparticles were required. To achieve such a concentration, CdSe-TOPwas precipitated and then resuspended in a minimal volume of analternative solvent. Tests for solubility of the precipitated CdSe-TOPnanoparticles were performed in three different solvents. Inmicrocentrifuge tubes, 300 μL of 100 μM CdSe-TOP nanoparticles wereprecipitated with the addition of 1.0 mL of ethanol and then pelleted bycentrifugation (5030×g, for five minutes). The CdSe-TOP pellet was thenresolubilized in 300 μL of three different solvents: NMF, acetone andethyl acetate. The CdSe-TOP nanoparticles were soluble in the NMF andthe ethyl acetate, but were insoluble in the acetone. NMF was chosen tobe the solvent for the EPR experiments since NMF is the best knownsolvent for stabilizing FeMo-co and it was observed to be miscible withchloroform.

To inspect if the CdSe-TOPFeMo-co complex would stay intact during theethanol precipitation and resolubilization to enable concentration ofthe sample, CdSe-TOP before and after mixing with FeMo-co werecharacterized by UV-vis absorbance and fluorescence emission.Concentrations of reagents were the same as used in the fluorescenceemission experiments. Ethanol was added to the CdSe-TOPFeMo-co sampleand was centrifuged at 5030×g for five minutes. The pellet wasresolubilized in chloroform and the fluorescence emission spectrumshowed no change from before the ethanol precipitation. The UV-visspectra also showed no changes, and thus the CdSe-TOPFeMo-co complexappeared to be intact after one ethanol wash.

Samples for EPR spectroscopy were prepared by mixing 3.0 mL CdSe-TOP(400 μM) with 0.67 mL FeMo-co (60 μM) and then precipitating with theaddition of 10.0 mL of ethanol with rapid mixing on a vortexer. Thesample was centrifuged (5030×g, for five minutes) to pellet theprecipitated CdSe-TOPFeMo-co. The pellet was solubilized by adding 250μL of solvent NMF solution and vortexed. The sample was injected into asealed anaerobic 4 mm quartz EPR tube and frozen in liquid nitrogen.

The three control samples were as follows. The first control, CdSe-TOP,showed no EPR signal (not shown). The second control was that of 180 μMMoFe protein (2 FeMo-co clusters per protein molecule), (FIG. 5, Trace1). The third control was that of 60 μM FeMo-co (FIG. 5, Trace 2) [41,42].

The EPR inspection of the CdSe-TOPFeMo-co (FIG. 5, Trace 3) showssignificant changes compared to the MoFe protein or isolated FeMo-co.First, the EPR signal at g=4.3, 3.7 and 2.0, representing the S=3/2 spinstate, is largely diminished and the S=½ spin state sub manifold signalhad a g value of 1.99. Second, a significant new signal at g=1.98 and1.97 is observed. Significantly, an EPR signal in this region of thespectra is from an S=½ spin state and suggests a change in electronicspin state, possibly due to reduction of the S=3/2 state of FeMo-co.Similar S=1/2 spin state signals have been observed with several trappedturnover intermediates of MoFe proteins and MoFe protein variants [43].Such intermediates are associated with turnover states that are relevantto the mechanisms of substrate reduction for various substrates. AnotherS=½ spin state signal has been observed in a trapped state during protonreduction by FeMo-co in a nitrogenase α-70^(IIe) MoFe protein variant[44]. Thus, the S=½ spin state in this experiment may be interpreted toindicate an electronic change in FeMo-co.

To further probe if the change in electronic spin state observed for theCdSe-TOPFeMo-co may indeed be due to light catalyzed electron transferfrom the CdSe, light dependent experiments were performed. FeMo-coshould appear in the resting state with its S=3/2 spin state signal ifthere would be no photo-initiated electron transfer from the CdSe-TOP.An additional sample was prepared as the CdSe-TOPFeMo-co sample in thelight; however, before being frozen in the EPR tube it was exposed toair. This sample would verify that the new S=½ EPR signal was indeed areduced state of FeMo-co and not Fe^(2+/3+) ions in solution fromdegraded FeMo-co. Preparation of these samples in the dark were met withtechnical difficulties and EPR inspection of these samples yielded nointerpretable data.

3.3 Assembly of CdSe-Mercaptosuccinic AcidNafYFeMo-co Complex

CdSe-TOP quantum dots in organic solvents are well known for theirphoto-activity, but the presence of an organic solvent limits their usewith most protein applications. Three approaches to achieve watersoluble CdSe quantum dots have been reported [45]. One is by a directsynthesis method which uses an arrested precipitation technique inaqueous media [46]. A second synthetic method encapsulates theorganically soluble quantum dot in a polymerized silica shellfunctionalized with polar groups or an amphiphilic polymer coating thatcan significantly add to the quantum dot diameter [47]. Third, a ligandexchange method replaces the capping agent on the nanoparticle fromtrioctylphosphine or trioctylphosphine oxide with a mercapto-carboxylicacid [48].

A disadvantage with water soluble nanoparticles is their tendency to beless photo-active than their organic counterparts. If there is a lack ofcoordinated surface atoms, then there will be trapped states on thesurface of the quantum dot that lie within the band gap. When lightilluminates the nanoparticle and excites electrons to the conductionband, there will be more alternative pathways available for relaxationback to the ground state via the trapped states [48]. Thus the desiredenergy or electron transfer to any adsorbed surface species may notoccur. The challenge was to determine the best consistent method toachieve the most photo-active aqueous soluble quantum dots.

Methods to produce the most luminescent quantum dot were exploredthrough two efforts to directly synthesize the quantum dots [49, 50],and by three methods to exchange the capping agent [29, 51, 52]. Cappingagents affect the photoluminescent properties of the nanoparticles. Themost luminescent quantum dots with a target diameter (2.4 to 2.6 nm)were produced by a published method with some modifications usingpreviously unused mercaptosuccinic acid (MSA), as the capping agent[29]. The previous complex between CdSe-TOP and FeMo-co took advantageof both reactants being in organic solvents. When isolated FeMo-co ismixed in water, even though kept anaerobic, the FeMo-co quicklyhydrolyzes. For FeMo-co to combine with aqueous CdSe and remain intact,complexation to proteins was required.

NafY is a low molecular weight protein that binds FeMo-co with highaffinity (K_(d) of 62 nM) [27]. Combining FeMo-co with NafY andconjugating this species to CdSe-MSA may be a useful way to place theFeMo-co in close proximity to the photo-reducing CdSe-MSA. TheNafYFeMo-co conjugated to CdSe in a Tris buffered system would be ameans for isolated FeMo-co bound to NafY to be in an aqueous system. The26 kDa NafYFeMo-co system has a further advantage, compared to the 250kDa MoFe protein that contains FeMo-co in a more buried configuration,of being smaller and of providing better accessibility to FeMo-co.Furthermore, the aqueous solvent would be a better proton source forhydrogen evolution.

Although aqueous CdSe quantum dots are less luminescent (by a factor of40), than CdSe in an organic solvent of the same diameter, thusindicating a lower quantum yield, their ability to be involved inelectron transfer was anticipated. Inspection of the CdSe-MSAnanoparticles was performed to determine their capability to formcomplexes and their photoactive capability to transfer electrons toFeMo-co and thereby perform interesting and useful chemistry.

To use CdSe-MSA in experiments with a FeMo-co species, demonstration ofCdSe-MSA forming a complex with proteins was necessary. CdSe-MSA wasadded in incremental amounts to bovine serum albumin (BSA) according toa published method and thereby demonstrating complex formation betweenthe two reagents [54]. The published procedure had used CdSe capped withmercaptoacetic acid instead of the mercaptosuccinic acid capping theCdSe in this study. The samples were excited at 280 and 410 nm in afluorescence emission investigation. Control samples of BSA alone andCdSe-MSA alone were excited at both wavelengths with no observablechanges seen in the samples.

Emission at 347 nm was from the BSA and emission from the 532 nm wasattributed to the CdSe-MSA. The BSA fluorescence emission was quenched78% with the addition of 300 μL of CdSe-MSA. The ratio of the twocomponents, BSA:CdSe-MSA, was 1:3.5. The conclusion was that CdSe-MSAand BSA protein had formed a complex.

To probe complex formation between CdSe-MSA and NafY protein, the twowere combined by adding incremental amounts of CdSe (14.6 μM stock) toNafY (500 nM) (FIGS. 6A and 6B). The sample was excited at 280 nm toobserve tryptophan emission at 347 nm. The sample was also excited at410 nm to verify that the observed peak at 532 nm could be attributed tothe CdSe-MSA being added in increasing amounts. Eighty percent of theNafY fluorescence quenching was observed with 30 μL addition ofCdSe-MSA, which is a 1:1 ratio between NafYCdSe-MSA (FIG. 6B). A NafYcontrol sample and a CdSe-MSA control sample showed no changes whenexcited at 280 nm and 410 nm. FIG. 6C shows the data from 6A replottedas percent quenching of NafY fluorescence versus concentration ofCdSe-MSA. FIG. 6D shows the data from FIG. 6B replotted as CdSe-MSAfluorescence emission versus concentration of CdSe-MSA.

Additionally, Forster resonance energy transfer (FRET), could beinterpreted from the fluorescence emission experiments. Two sets ofemissions (347 nm and 532 nm) were observed (FIG. 7). Emission at 347 nmis from excitation of the tryptophans in NafY protein that acts as adonor for CdSe-MSA, which emits at 532 nm. The first emission at 347 nm(rust brown trace) shows NafY before addition of CdSe-MSA. The secondemission (forest green trace) exhibits quenching of NafY fluorescencealong with an acceptor emission at 532 nm from CdSe-MSA demonstratingFRET. When the sample was excited at 410 nm, the emission at 532overlapped the emission observed with excitation at 280 nm confirmingthat the 532 nm emission was indeed from CdSe-MSA. FRET emission canonly be observed when two species are close in proximity hence theconclusion was that NafY and CdSe-MSA had formed a complex.

To address the issue of lesser photo-activity of the water solublequantum dots as compared to the organically solvated nanoparticles, anexperiment with aqueous CdSe-MSA nanoparticles was performed. Thehypothesis was that despite the photo-activity being less, the watersoluble CdSe-MSA quantum dots would be able to perform a photo-inducedelectron transfer to a complexed species adsorbed onto their surface. Totest the photocatalytic reduction activity, methyl viologen was combinedwith and CdSe-MSA. Dithiothreitol (DTT) would be used as a sacrificialelectron donor to refill electron holes generated after electrons wereexcited to the conduction band from the valence band in CdSe-MSA.Reduction of the methyl viologen, MV²⁺, was monitored by increasingabsorbance peaks at 395 nm and at 603 nm with increasing times ofillumination.

CdSe-MSA (720 nM), methyl viologen (1.47 mM) and dithiothreitol (14.7mM), were combined and monitored by UV-vis absorbance. The sample wasilluminated for 30 seconds and followed by UV-vis absorbance. Thisprocess was repeated for a total of eight minutes. To further test thephotocatalytic ability of the system, the sample was first exposed toair to oxidize the reduced methyl viologen. Next, the cuvette wasresealed and put on the manifold to make the headspace anaerobic byreplacing it with argon. The photo-induced reduction experiment wasrepeated. Another air oxidation was followed by a third repetition ofthe experimental procedure. The first control sample was a solution ofmethyl viologen and dithiothreitol which underwent the same illuminationtimes and UV-vis absorbance characterization. The second control was theCdSe-MSA for which the same experimental procedure was followed. A thirdsample was set up with the three reactants and kept in the dark for 16hours.

The results show increasing absorbance at both 395 nm and at 603 nm withincreasing periods of illumination (FIG. 8A). The data for theabsorbance at 603 nm was replotted as absorbance intensity versus timeof illumination (FIG. 8B). After exposing the sample to air andrepeating the illumination and UV-vis absorbance measurements, thesample again demonstrated increasing absorbance at 395 and 603 nm withincreasing periods of illumination. Interestingly, the CdSe-MSA-methylviologen solution turned progressively more intense in blue color withincreasing periods of illumination, indicative of absorption at 603 nm.When the sample was exposed to air, it lost the blue color and wasorange from the CdSe.

When the second round of illumination progressed the blue color returnedto the sample. When the third repetition was performed, the same resultswere observed. The three controls showed no increasing absorbance at 395nm or 603 nm with illumination. Additionally a concentration dependentexperiment was performed with twice the CdSe-MSA (1.4 mM). The resultsshowed that the absorbance peak at 603 nm increased more rapidly withthe higher concentration of CdSe-MSA and thus showed a general trend ofconcentration dependence in the photo-reduction of methyl viologen (FIG.8B). The conclusion was that the CdSe-MSA quantum dots were photoactiveand able to perform photo-reduction of adsorbed species, MV²⁺ in thiscase, when illuminated with intense visible light.

3.4 EPR Characterization of CdSe-MSANafYFeMo-co

Experiments above present evidence that the CdSe-MSA will complex withthe NafY protein and that CdSe-MSA is able to photo-reduce a speciesadsorbed onto its surface upon exposure to visible light. Thus,NafYFeMo-co being close in proximity to CdSe-MSA should allow photocatalyzed reduction and possible post-illumination changes in theelectronic structure of FeMo-co that could be observed by EPRspectroscopy.

NafYFeMo-co samples were prepared prior to forming a complex with theCdSe-MSA. NafY protein was diluted to approximately 1.0 M in 25 mM Tris,pH 8.0, 0.5 mM sodium dithionite in a stirred ultrafiltration cellfitted with a YM10 membrane (Millipore). To prevent precipitation ofNafY, additions of FeMo-co to the stirred cell ultrafiltration devicecontaining NafY were made such that the NMF, solvating FeMo-co, did notexceed 3% (v/v). Repeated concentration and dilution steps wereperformed with each addition of FeMo-co. After the last addition ofFeMo-co, 1.5 mL of degassed CdSe-MSA (30 μM) with 0.4 mM sodiumdithionite was added. The solution was concentrated further to minimizethe volume and then transferred to a 30,000 MWCO Centricon (Millipore)and kept anaerobic in JA-20 centrifuge tubes. Centrifugation wasperformed at a centrifugal force of 5000×g to reach the desiredconcentration. The sample was then injected into 4 mm EPR quartz tubesand frozen in liquid nitrogen. The final concentration of the sample was400 μM CdSe-MSA, 200 μM NafYFeMo-co.

Control samples were as follows. The first was a FeMo-co sample used tocomprise the CdSe-MSANafYFeMo-co samples. The second control was aNafYFeMo-co sample. The third control was the CdSe-MSANafYFeMo-cosample prepared as previously described under strict anaerobicconditions throughout, but before being frozen it was exposed to air.With air exposure, FeMo-co should oxidatively degrade, resulting inferrous and/or ferric ions in solution. This control sample woulddemonstrate that any change seen with the FeMo-co signal, did not arisefrom dissolved iron in solution. One last control sample was that ofCdSe-MSA alone at a concentration of 400 μM.

The FeMo-co control spectrum showed an EPR signal (g=4.65, 3.44 and1.97) for an S=3/2 spin state, characteristic of its dithionite reducedstate when extracted into solvent NMF solution (FIG. 9, Trace 1) [42].Compared to the MoFe protein, with FeMo-co in the active site, the EPRspectrum for extracted FeMo-co was of a similar line shape, onlybroader. There were also comparable principal g-values, indicating thatthe general electronic structure was coincident. Binding of FeMo-co toNafY as in the NafYFeMo-co yields a similar spectrum with g=4.41, 3.71and 2.07 (FIG. 9, Trace 2), indicative of an S=3/2 spin state [27]. Thebinding of NafY does sharpen the line shape and is consistent with anadditional ligand to FeMo-co, indicating covalent bonding between NafYand FeMo-co.

EPR characterization of the CdSe-MSANafYFeMo-co sample (FIG. 9, Trace3) showed an EPR signal for the S=3/2 spin state, very similar to thatof NafYFeMo-co, significantly indicating that the electronic structureof FeMo-co bound to NafY was not being perturbed when the protein wasinteracting with the CdSe. There was a change in the g˜2 portion of thesignal, corresponding to the S=½ submanifold, but the rest of the signal(g=4.41 and 3.71) remained unchanged. Hence, the structural integrity ofFeMo-co, both molecular and electronic, should not be affected in thiscomplex. This meant that FeMo-co survived the process and procedure forformation of the CdSe-MSAFeMo-co ternary complex and would be suitablefor photo-reduction.

The effect of light on the CdSe-MSANafYFeMo-co sample was examined.The sample was sealed and thawed to room temperature anaerobically. Thesample was exposed to a high intensity light for ten seconds. The samplewas then immediately frozen in a hexanes-liquid nitrogen slush bath andre-characterized by EPR. The EPR spectrum (FIG. 10, Trace 2), showedsignificant change as compared to this same sample's previous EPRspectrum (FIG. 10, Trace 1, duplicate of FIG. 9, Trace 3). The postilluminated sample shows a largely diminished EPR intensity for aspecies with the same line shape as the S=3/2 state prior toillumination. The diminishment in signal intensity corresponds to achange in the population of S=3/2 spin state to some other EPR silentstate, suggesting that illumination initiated an electron transfer fromCdSe-MSA to the adsorbed NafYFeMo-co and therefore reducing it. Thisilluminated sample was then rethawed again, allowed to air oxidize, andrefrozen. EPR inspection of the sample after air exposure (FIG. 10,Trace 3), shows a sharp signal at g=2.0, clearly from ferric irons.Thus, the conclusion was that upon exposure to intense light, theelectronic state of FeMo-co changed due to a possible electron transferfrom the CdSe-MSA to the NafYFeMo-co.

3.5 Photocatalyzed Hydrogen Generation Experiments

The experiments presented above demonstrate that the CdSe-MSA would forma complex with the NafY protein. The EPR based characterization of theCdSe-MSANafYFeMo-co system showed that photo-reduction of FeMo-co uponillumination was likely. The CdSe-MSA had exhibited photo-reductiveactivity by its ability to reduce methyl viologen when illuminated witha 500 watt halogen lamp, detected by changes in UV-vis absorbance. Allof these evidences supported the hypothesis that a CdSe-MSANafYFeMo-cosystem when illuminated should perform a reduction of aqueous protons asschematically described in FIG. 11. Sodium dithionite (2 mM) would beadded to the system for the dual purpose of 1) acting as a sacrificialelectron donor to refill the electron hole after the generation ofexcitons in the CdSe upon illumination, and 2) acting as an oxygenscavenger to preserve the anaerobic environment for FeMo-co.

The hydrogen generation experiments were performed with the variablebeing the extent of excess of CdSe-MSA with respect to NafYFeMo-co,ranging in ratios of 8:1, 4:1, 2:1, 1:1 and 0.5:1. The NafYFeMo-co wasprepared as previously described with all the NafY bound with FeMo-co ina 1:1 ratio. The CdSe-MSA and NafYFeMo-co were initially mixed with alow concentration of sodium dithionite (0.2 mM) and allowed to mix forone minute to form a complex without interference from sodiumdithionite. After the initial time for complex formation, additionalsodium dithionite was added to a final concentration of 2.0 mM. Allsamples and controls were then positioned in front of the halogen lamp.

The head space gas of experimental and control samples were analyzed atmultiple time points by gas chromatography. Samples from controlreactions of CdSe-MSA, NafYFeMo-co and CdSe-MSAFeMo-co showed no signsof hydrogen throughout. Interestingly, no hydrogen was initiallydetected, but hydrogen was detected after 24 hours of illumination fromthe reaction with 1:1 ratio of CdSe-MSA to NafYFeMo-co. The othercomparable reactions with other ratios of CdSe-MSANafYFeMo-co showedno signs of hydrogen generation. The experiment was repeated and stillonly the 1:1 samples showed hydrogen production. All total there werefour different repetitions of the reaction with 1:1CdSe-MSANafYFeMo-co that generated hydrogen starting at 24 to 30 hoursand continuing to produce hydrogen until at least 114 hours (FIG. 12A).

Two of the 1:1 samples were duplicates with samples analyzed at the sametime and the data are shown in FIG. 12B with error bars to demonstratereasonable consistency. The generation of hydrogen continued to between100 to 114 hours. The maximum amount detected at that point was 316nmols of hydrogen produced over a period of 64 hours. With respect tothe amount of FeMo-co used in the reaction, 105 nmol H₂ was produced pernanomole of FeMo-co. Stated as a specific rate, 1.8 nmol H₂/nmolFeMo-co/hour was the rate of hydrogen production.

The hydrogen generation experiments were also attempted with MoFeprotein in 100 mM MOPS, 1 mM dithionite, pH of 7.0 with added CdSs-MSA.Previously, a report [7], has shown the evolution of hydrogen from MoFeprotein with Ru(bypy)₂ attached.

As in the experiment above with the CdSe-MSANafYFeMo-co, reactionswith CdSe-MSA and the MoFe protein in varying ratios were set up.Control samples were CdSe-MSA (720 nM) and MoFe protein (360 nM). Headgas samples (100 μL) were analyzed by gas chromatography.

Initially, precipitation was observed with the higher ratios of CdSe-MSAto MoFe protein within the first 30 minutes of illumination. The 2 to 1sample remained soluble and translucent for three hours but becamecloudy thereafter. Solubility experiments were performed with differentpH buffered solutions of 7.0 to 8.0. The conditions with pH of 7.8 and8.0 showed better solubility with the CdSe-MSAMoFe protein system ascompared to the lower pH values. No notable hydrogen production wasobserved with these set of samples.

An additional reaction was performed with CdSe-TOP and FeMo-co. Giventhe high photo-activity of the CdSe-TOP, a sample in chloroform wascombined with FeMo-co dissolved in NMF solution with all solutions andsamples thoroughly degassed. The reaction was performed in a quartzcuvette with 5.0 μM CdSe-TOP and 1.25 μM FeMo-co. The headspace wasanalyzed after one hour of illumination and determined to contain 28nmol of hydrogen. This reaction was performed lacking controls and needsto be re-performed in a proper experimental context with appropriatecontrols and variables. Although this result needs to be furtherinvestigated, the result is noteworthy due to its relative highactivity. This system should be investigated further with duplicates,concentration dependencies and control samples to corroborate thisresult.

4. Discussion

Toward the goal of producing hydrogen passively as an alternative fuel,a novel advanced material has been developed. CdSe nanoparticlescomplexed with FeMo-co, in both aqueous and organic solvent systems wereilluminated with visible light and evolved hydrogen. TheCdSe-MSANafYFeMo-co system when illuminated evolved hydrogenconsistently in four different experimental sets under the same reactionconditions. The CdSe-TOPFeMo-co system produced hydrogen in one sample,but awaits optimization. While not wishing to be bound by theory, therationale behind the experiments was as follows. CdSe nanoparticles withbandgap energy of 1.7 eV are able to absorb incident light in thevisible range, and create an electron-hole pair. CdSe nanoparticles wereselected because their band gap energy is a close match to thethermodynamic potential of the H⁺/H₂ couple. With the addition ofNafYFeMo-co to the CdSe nanoparticles, a complex was formed and thusput the NafYFeMo-co in close enough proximity so that the excitonparticipates in electron transfer to the NafYFeMo-co. Thisphoto-induced reduction of FeMo-co then in turn catalyzed the reductionof protons in the aqueous solution to form hydrogen.

4.1 Efficacy of Hydrogen Generation from CdSeFeMo-co Systems

There were some important discoveries along the way that contributed tothe success of assembling a complex that when exposed to high intensitylight would result in hydrogen evolution. Utilization of CdSe-TOP andthe CdSe-MSA nanoparticles with sizes that matched the bandgap energyassociated with the thermodynamic potential of the H⁺/H₂ couple and theadsorbed FeMo-co's reduction potential to facilitate its photo-reductionwas crucial. CdSe nanoparticle sizes in the range of 2.4 to 2.7 nm indiameter appeared to be suitable for complex formation, monitored byfluorescence emission quenching and EPR. Particles of this size alsowere suitable for hydrogen experiments.

Electron transfer between the excited CdSe nanoparticles and theadsorbed FeMo-co species was necessary to reduce FeMo-co. For this tooccur, a complex had to form between CdSe and FeMo-co. This wasdemonstrated both in the organic CdSe-TOPFeMo-co system and the aqueousCdSe-MSANafYFeMo-co system by fluorescence emission quenching. Toobtain evidence that in both complexed systems FeMo-co would bephoto-reduced, samples were interrogated by EPR spectroscopy. The EPRspectrum with the CdSe-TOPFeMo-co system indicated that the populationof the resting state S=3/2 spin state was largely diminished. A newsignal was observed at g=1.98 and 1.97. This observed S=½ spin statesuggested a change in electronic spin state, possibly due to reductionof the S=3/2 spin state of FeMo-co.

The EPR spectra of the CdSe-MSANafYFeMo-co system showed a similarS=3/2 spin state with a sharpened line shape consistent with anadditional ligand (NafY) on FeMo-co. The complexed system spectrum wassimilar to the NafYFeMo-co control sample indicating that theinteraction between CdSe-TOP and FeMo-co had not perturbed the FeMo-coelectronic structure. FeMo-co had survived the complex formation processand would be suitable for photo-reduction.

When the effect of light on this same CdSe-MSANafYFeMo-co sample wasexamined, the EPR spectrum showed a significant change in the S=3/2 spinstate of FeMo-co compared to the previous EPR spectrum. The postilluminated sample showed a largely diminished EPR species with the sameline shape as the S=3/2 spin state prior to illumination. The diminishedsignal intensity corresponds to a change in the population of the S=3/2spin state suggesting that illumination had initiated an electrontransfer from CdSe-MSA to the adsorbed NafYFeMo-co.

The most successful system of the aqueous and organic systems withrepeatable results for hydrogen generation was a one to oneCdSe-MSANafYFeMo-co system. The maximum hydrogen production was 316nanomoles or 105 nanomoles H₂/nanomoles NafYFeMo-co. There was oneCdSe-TOPFeMo-co sample which was illuminated and produced 28 nanomolesof hydrogen in one hour; 22.4 nanomoles H₂/nanomoles FeMo-co. Theexperiment with the CdSe-TOPFeMo-co sample needs to be re-performed ina proper experimental context with appropriate controls and variables.

There were other considerations that are beneficial for analyzing thestudy. The interaction between the CdSe nanoparticles and the FeMo-cowas different in the organic versus aqueous systems. The CdSe-TOP andthe FeMo-co formed a complex in a direct interaction between the two.This resulted in an EPR signal at g=1.99 resulting in an S=½ spin statesub-manifold signal. Coordination of Fe in FeMo-co to the selenium ofthe nanoparticle may have produced this change in electronic structureof the FeMo-co [39].

In the aqueous system, the CdSe-MSA forms a complex with the NafYprotein. Because of the size of NafY being approximately 15 nanometersin diameter and the FeMo-co bound to the NafY being 8 to 10 angstroms(0.8 to 1.0 nm), this resulted in FeMo-co being close enough forelectron transfer without being directly complexed with thenanoparticle. This was shown in the EPR spectrum of theCdSe-MSANafYFeMo-co system when first combined. There was littlechange in the EPR signal at g=(4.41, 3.71 and 2.03) representing theS=3/2 spin state; however, when this sample was illuminated with intensevisible light, the EPR signal intensity was largely diminishedindicating a change in the population of S=3/2 spin states to somecorresponding EPR silent state. An EPR silent signal is indicative of adiminished concentration of unpaired spins [55].

This change was interpreted to mean that electron transfer had occurredbetween the excited CdSe-MSA to the FeMo-co inside NafY.

A few other factors to consider in the analysis of what led to thehydrogen evolution are as follows. There is a distinct advantage of theaqueous based CdSe-MSANafYFeMo-co system because of the ready sourceof protons as opposed to the chloroform dissolved CdSe-TOPFeMo-co. Thesamples were set 8 centimeters from the 500 watt halogen lamp, whichpromoted catalysis. Temperature maintenance was important in order toprevent degradation of the NafY protein conjugated to FeMo-co, 30±2° C.FeMo-co itself is not as temperature sensitive as the protein.

4.2 Possible Explanation for the Observed Lag of theCdSe-MSANafYFeMo-co System for Hydrogen Generation

One observed phenomenon in the hydrogen generation experiments using theCdSe-MSANafYFeMo-co system was interesting. The system did not showdetectable hydrogen until about 24 hours after the initiation ofillumination. The most plausible explanation for the observed lag couldbe that there were pH effects from dithionite reacting with wateraccording to equations 2 and 3 [56]. Over time the pH would decrease andmore protons would be available for reduction, especially as theequilibrium shifted right as shown in equation 2. The sodium dithioniteis in excess in the reaction vessel. To explain the eventual cessationof hydrogen production, once the dithionite was depleted then thehydrogen production levels off because protons are less available. Anexperiment interrogating the effects of pH on hydrogen production eitherby direct changes in the pH of the buffered solution and/or changes insodium dithionite concentration would be beneficial towards probing thisissue.

S₂O₄ ²⁻

2SO₂ ⁻(I  (Eq. 2)

SO₂ ⁻+H₂O

HSO₃ ⁻+H⁺ +e ⁻(I  (Eq. 3)

The reaction in equation 2 is shown as an equilibrium between the dimerand monomer forms of dithionite. It has been shown that the realelectron donating species is the radical monomer, SO₂ ⁻ [59]. As statedpreviously, the dithionite serves as an electron donor to fill holesgenerated in the valence band with CdSe exciton formation. The secondpurpose of dithionite is to serve as an oxygen scavenger to preserveanaerobicity in the reaction vessel. An additional purpose may be toindirectly supply protons for reduction. So reaction conditions thatwould favor the formation of the radical monomer species and therebyincrease the H+ concentration as it reacted with water would facilitatethe overall reaction of hydrogen generation.

The experimental setup required a cooling method to maintain atemperature of 30° C. to prevent degradation of the NafY protein in thereaction vessel. This required the use of a Peltier cooling devicebecause the halogen lamp used readily heated the test reaction vesselcontents to 55° C. and above. Reaching a stable temperature in thereaction vessel may have taken a number of hours. Higher temperatures,tested in the range of 2 to 40° C., have shown a tendency to shift thedithionite equilibrium to the monomer side [59]. The NafY protein didprove to be more robust than our expectations. Experiments could bedesigned to find the maximum hydrogen generation corresponding to ahigher temperature. Exploration of pH effects and increasedconcentrations of dithionite could also enhance the monomer side of theequilibrium reaction and merit investigation [59].

Other variables that could be explored to reduce the lag time could beincreasing the intensity of the light and varying the concentrations ofthe reactants. The explanation for the lag in H₂ production deservesfurther experimentation with the proper controls and variables.

4.3 Comparison of this Hydrogen Production Method with Other PublishedMethods

The generation of hydrogen from a sophisticated bioconjugate materialsuch as CdSe-MSANafYFeMo-co is significant. To gauge how significant,the effectiveness of the system presented as part of the current studyis compared to other published bioconjugate hydrogen generation methods(Table 2). The amount of hydrogen or hydrocarbon production and therates for the hydrogen formation have been recalculated for directcomparisons. The first method combines CdSe nanoparticles withhydrogenase at a pH of 4.75 with ascorbic acid being the electron donorthat produced hydrogen at an impressive rate (93 nmol H₂/nmol activematerial/min). However, the reaction was only able to produce hydrogenat this rate for five minutes, possibly due to degradation of theprotein with the acidic conditions [5]. The second method involvedseveral MoFe proteins variants that allowed for conjugation of aRu(bypy)₂ photosensitizer to one of three substituted cysteines near thebound FeMo-co. The system was able to produce 1.9 nmol H₂/nmol activematerial/min. The electron donor used was 200 mM dithionite and it wasnecessary that this excess be used to sustain the reaction for 50minutes, which diminished in production after that point [7].

TABLE 2 Analytical Comparison of Different Hydrogen Production Systems*Rate of Production (H₂/nmol active Maximum Time Ref. Reaction Methodmaterial/minute) (nmol) (min) No. Conditions CdTe/Hydrogenase 93 70 5 5pH 4.75 Ru(bypy)₂/MoFe protein 1.9 2300 50 7  200 mM Na₂S₂O₄**Eu^(II)-DTPA/FeMo-co 0.097 5.8 60 57 CdSe-MSA-NafY-FeMo-co 0.030 3166840 this pH 8.0; study  2.0 mM Na₂S₂O₄ *Rates of production have beenrestated in equivalent units for comparison purposes. **Ethyleneproduction measured

Another method of not hydrogen production, but hydrocarbons production,is included since it uses FeMo-co. FeMo-co was combined with cyanide ionand europium (II) diethylenetriaminepentaacetate [Eu^(II)-DTPA], astrong photoactivated reductant. Hydrocarbons were produced with thissystem and more particularly ethane at 0.097/nmol H₂/nmol activematerial/min. The reaction was also attempted with carbon monoxide, butthe hydrocarbons produced were considerably less. FeMo-co typicallyhydrolyzes in aqueous solvents, however, in this report it showed 85%activity after the first hour of being in an aqueous based solvent [57].

As seen in Table 2, the effectiveness of the CdSe-MSANafYFeMo-cosystem for hydrogen formation is comparable with the other reportedsystems. In terms of the rate of production, this system requiresimprovement to be competitive. However, this system is competitiveenough, so that efforts expended towards increasing the amount producedand the rate of production are worthwhile. In terms of stability, theCdSe-MSANafYFeMo-co system may have an advantage in that it wasoperational for 90 plus hours versus five minutes or one hour or fourhours as with the other published systems. The length of time that thesystem is catalytically active could be a real plus as long asoptimization of the system could increase the rate of hydrogenproduction.

4.4 Potential Improvements for the CdSeFeMo-co Systems

Ways to enhance the hydrogen production using the CdSe-MSANafYFeMo-cosystem include the following. The current systems were examined at a pHof 8.0. In the aqueous CdSe-MSANafYFeMo-co system, a feasibleexperiment would be to probe the limits of pH with respect to hydrogenproduction, the stability of the system, and maintaining the solubilityof the system. At lower pH values, the higher concentration of protonsas substrates will drive the equilibrium forward. The method usingCdTe-hydrogenase discussed earlier utilized a pH of 4.75 [5]. Impressivehydrogen production was observed, but only for five minutes. pH alsoplays a role in maintaining solubility with the CdSe-MSA. If themercaptosuccinic acid is protonated, the result may be that thenanoparticles lose their ability to be aqueously solubilized. ThepK_(a)s of the carboxylic acid functional groups in mercaptosuccinicacid are pK_(a1) of 3.64 and pK_(a2) of 4.64. Consequently, any bufferedsolution in the pH range of 7.0 to 8.0 used to form the complex shouldmaintain solubility. As seen with CdSe-MSAMoFe protein experiments, pHlower than 7.8 decreased the solubility of the system. There could havebeen more non-specific binding between the MoFe protein and thenanoparticle that could have contributed to diminished solubility. Astudy comparing solubility of the system compared to the protein withthe FeMo-co (MoFe protein versus NafY protein), complexed to theCdSe-MSA would be beneficial to determine the size of the protein andits effects on solubility.

As mentioned earlier, the effects of decreasing pH from the presence ofsodium dithionite and its hydrolysis could be a variable pertaining tothe molar volume of hydrogen produced. The effects of different sodiumdithionite concentrations could be investigated. With an increase in thesodium dithionite, it will be important to combine the CdSe-MSA andNafYFeMo-co at a lower concentration (0.2 sodium dithionite) as wasdone previously. This will give the complex adequate time to avoidcompeting effects from the dithionite for positions on the CdSe-MSAsurface. A validation that higher dithionite concentration may increasethe amount of hydrogen produced is found in the report that utilized theMoFe protein with bound Ru(bypy)₂. In that study, higher dithioniteconcentration, in a range from 20 mM to 200 mM, resulted in greateramounts of evolved hydrogen corresponding to increased dithionite [7].The reaction with 200 mM dithionite produced a fivefold higher amount ofhydrogen produced compared to the 20 mM dithionite sample. Anotherconsideration regarding the dithionite is the temperature dependentequilibrium favoring the monomer form of dithionite which is thereducing species [59]. The system was more robust than anticipated andcould possibly withstand higher temperatures.

There are other experimental conditions or variables that could beinvestigated in order to optimize hydrogen production. A different kindof nanoparticle could be synthesized and used as in the previousexperiments such as cadmium telluride (CdTe). The bandgap energy of CdTemay be a closer match for the H₂/H⁺ thermodynamic couple and FeMo-co'sreduction potential. A study with various size of the same kind ofnanoparticle could be conducted in order to ascertain any size dependentresults for hydrogen production. Other variables to explore in thehydrogen generation experiments would be the kind of lamp used forillumination and temperature effects on the hydrogen generation.

An interesting issue is the source of protons with the organicCdSe-TOPFeMo-co system in chloroform. The 1 mM dithionite, 25 mM Trissolution in NMF solution with FeMo-co is the source of protons. With theaddition of 5 μL of FeMo-co in NMF, 1 mM dithionite this equates tointroducing 100 nanoliters of water to the sample. The one cuvettesample saw the production of 28 nmols of hydrogen in one hour. How bestto increase the source of protons in this system warrants furtherexperimentation. The addition of pentafluorothiophenol (C₆F₅S⁻) toFeMo-co was suggested as a source of protons catalytically, not as anultimate source [57]. The C₆F₅S⁻ replaces one of the amide ligands ofthe solvent NMF solution. Although the use of this ligand wasinadequately explored in the study referenced, it does deserveinvestigation.

5. Conclusion

This study described complexes formed between CdSe and FeMo-co inorganic and in aqueous solvents and the experiments probing theirinteractions, along with their capability to produce dihyrogen whenilluminated. While the experimental system studied here requires furtheroptimization, it contributes to the pursuit of developing sustainableenergy sources.

Various aspects of the present disclosure may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

The phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

INCORPORATION BY REFERENCE

All publications, patents and patent applications cited above areincorporated by reference herein in their entirety for all purposes tothe same extent as if each individual publication or patent applicationwere specifically indicated to be so incorporated by reference.

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1. A method for preparing a system for photocatalytically producinghydrogen gas, comprising: (a) providing a water soluble cadmium selenidenanoparticle (CdSe) surface capped with mercaptosuccinate (CdSe-MSA);(b) providing a NafYFeMo-co complex comprising a NafY protein and aniron-molybdenum cofactor (FeMo-co); and (c) mixing the CdSe-MSA and theNafYFeMo-co complex under anaerobic conditions to form aCdSe-MSANafYFeMo-co system, wherein when illuminated with a visiblelight source, the CdSe-MSANafYFeMo-co system is capable ofphotocatalytically producing hydrogen gas.
 2. The method of claim 1,wherein step (a) further comprises exchanging surface capping agent fromtrioctylphosphine (TOP) in a CdSe-TOP nanoparticle to mercaptosuccinate,to form the CdSe-MSA.
 3. The method of claim 2, wherein the CdSe-TOPnanoparticle has a diameter of about 2.4 nm to about 2.7 nm and theCdSe-MSA has a diameter of about 2.6 nm.
 4. The method of claim 2,wherein the exchanging step is performed in methanol under reflux in thepresence of a base.
 5. The method of claim 4, wherein the base istetrabutylammonium hydroxide.
 6. The method of claim 1, wherein in step(b), the NafY protein is derived from Azotobacter vinelandii.
 7. Themethod of claim 1, wherein in step (b), the FeMo-co is derived from amolybdenum-iron protein (MoFe protein).
 8. The method of claim 7,wherein the MoFe protein is derived from Azotobacter vinelandii strainDJ995.
 9. The method of claim 1, wherein step (b) further comprisescombining the NafY protein with stepwise aliquots of the FeMo-co to formthe NafYFeMo-co complex.
 10. The method of claim 9, wherein the FeMo-cois provided in N-methylformamide (NMF) solution and added stepwise tothe NafY protein so that NMF does not exceed about 3% (v/v).
 11. Themethod of claim 1, wherein in step (c), the CdSe-MSA and theNafYFeMo-co complex are provided at about 1:1 molar ratio.
 12. Themethod of claim 1, wherein in step (c), the CdSe-MSA is provided in thepresence of a dithionite salt.
 13. The method of claim 12, wherein thedithionite salt is kept at a sufficiently low concentration so as toallow the CdSe-MSA and the NafYFeMo-co complex to bind to each other.14. The method of claim 13, further comprising increasing the dithionitesalt concentration to facilitate hydrogen gas production by theCdSe-MSANafYFeMo-co system.
 15. A method for producing hydrogen gas,comprising illuminating a CdSe-MSANafYFeMo-co system with a visiblelight source, wherein the system produces hydrogen gas for an extendedperiod of at least 5 hours.
 16. The method of claim 15, wherein thesystem produces hydrogen gas for at least 10 hours.
 17. The method ofclaim 15, wherein the system produces hydrogen gas for at least 90hours.
 18. A system for photocatalytically producing hydrogen gas,comprising: a water soluble cadmium selenide nanoparticle (CdSe) surfacecapped with mercaptosuccinate (CdSe-MSA); and a NafYFeMo-co complexcomprising a NafY protein and an iron-molybdenum cofactor (FeMo-co). 19.The system of claim 18, wherein the CdSe-MSA and the NafYFeMo-cocomplex are present in about 1:1 molar ratio.
 20. The system of claim18, wherein when illuminated with a visible light source, the system iscapable of photocatalytically producing hydrogen gas for an extendedperiod of at least 5 hours.
 21. The system of claim 18, wherein thesystem produces hydrogen gas for at least 10 hours.
 22. The system ofclaim 18, wherein the system produces hydrogen gas for at least 90hours.
 23. The system of claim 18, wherein the system is kept underanaerobic conditions.